GSM8002831: scRNA-seq (Smartseq2) of D2 Spontaneous Blastoids - cell 36; Homo sapiens; RNA-Seq
For bulk RNA-seq, total RNAs were collected and isolated from cells using RNAiso Plus (Takara). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using NEBNextUltra RNA Library Prep Kit for Illumina following manufacturer's recommendations. For scRNA-seq, CI-hnESCs and day 2 aggregates were dissociated and sorted by flow cytometer for Smart-seq2 library construction and the libraries were sequenced on MGISEQ2000 (MGI Tech) platform to generate 100-bp paired-end reads. Day 3, day6 blastoids and the blastoids upon removing MEK/BRAF inhibition from Aggrewell were manually picked and polled with a mouth pipette, washed once with DPBS and then dissociated with 0.25% trypsin-EDTA (Gibco) at 37 °C in 20%O2 and 5% CO2 incubator for 30 min. Attached cultured bE14 blastoids were first rinsed once with DPBS and then treated with cold DPBS and incubated in 4°C for 10-20min to allow breaking of the matrix. When the samples became mobile under a microscope upon gentle shaking, the bE14 blastoids were transferred to a clean tube using a cut-tip and dissociated with 0.25% trypsin-EDTA at 37 °C in 20%O2 and 5% CO2 incubator for 30 min. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). RNA libraries were prepared for sequencing using standard Illumina protocols